Methods for separating frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
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1. A method of producing a frozen-thawed sorted artificial insemination sample comprising:
- thawing frozen sperm cells;
staining said thawed sperm cells with a concentration of a Hoechst 33342 greater than 40 micromolar;
establishing the staining temperature between about 30°
C. and about 40 C.°
;
determining a sex characteristic of said sperm cells;
separating said sperm cells according to the determination of their sex characteristic;
isolating sperm cells separated according to the determination of their sex characteristic in a collection element at a rate of greater than 1000 sperm per second for either X-chromosome bearing sperm or Y-chromosome bearing sperm;
establishing a frozen-thawed sorted artificial insemination sample from said sperm cells isolated in said collection element; and
fertilizing an egg with said frozen-thawed sorted artificial insemination sample at success levels of at least about 70% of the success levels with sperm cells that have not been separated and/or frozen.
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Abstract
Devices, compositions, and methods for handling, separating, packaging, and utilization of spermatozoa (1) that can be derived from previously frozen sperm samples collected from a male mammal. Specifically, techniques to uniformily stain (2) spermatozoal DNA even when derived from previously frozen sperm and separation techniques to separate and isolate spermatozoa even when derived from previously frozen sperm samples into X-chromosome bearing and Y-chromosome bearing populations having high purity.
555 Citations
16 Claims
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1. A method of producing a frozen-thawed sorted artificial insemination sample comprising:
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thawing frozen sperm cells; staining said thawed sperm cells with a concentration of a Hoechst 33342 greater than 40 micromolar; establishing the staining temperature between about 30°
C. and about 40 C.°
;determining a sex characteristic of said sperm cells; separating said sperm cells according to the determination of their sex characteristic; isolating sperm cells separated according to the determination of their sex characteristic in a collection element at a rate of greater than 1000 sperm per second for either X-chromosome bearing sperm or Y-chromosome bearing sperm; establishing a frozen-thawed sorted artificial insemination sample from said sperm cells isolated in said collection element; and fertilizing an egg with said frozen-thawed sorted artificial insemination sample at success levels of at least about 70% of the success levels with sperm cells that have not been separated and/or frozen. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of producing a frozen-thawed sorted sperm sample comprising:
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thawing frozen sperm cells; staining said thawed sperm cells with a concentration of Hoechst 33342 greater than 40 micromolar for a period of time sufficient to achieve uniform staining; establishing the staining temperature between about 30°
C. and about 40 C.°
;determining a sex characteristic of said sperm cells; separating said sperm cells according to the determination of their sex characteristic; isolating sperm cells separated according to the determination of their sex characteristic in a collection element; and establishing a frozen-thawed sorted sperm sample from said sperm cells isolated in said collection element, the frozen-thawed sorted sperm sample being capable of fertilizing an egg at success levels of at least about 70% of the success levels with sperm cells that have not been separated and/or frozen.
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Specification